Drug preparations of reduced toxicity

ABSTRACT

Preparations of drugs in admixture with certain ligands are described which, when administered to animals or humans, are less toxic than conventional drug preparations. Although the toxicity of the drug-ligand preparations described is greatly reduced, the drug retains pharmacological activity.

This application is a continuation application of copending U.S. patentapplication Ser. No. 844,248 filed Mar. 24, 1986 now U.S. Pat. No.4,897,384, which is in turn a continuation of U.S. patent applicationSer. No. 604,503, filed May 2, 1984 now abandoned, which is in turn acontinuation-in-part of U.S. patent application Ser. No. 498,268 filedMay 26, 1983, now abandoned.

TABLE OF CONTENTS

1. Field of the Invention

2. Background of the Invention

2.1. The Aminoglycoside Antibiotics

2.2. The Polyene and Polyene Macrolide Antibiotics

2.3. Adriamycin

2.4. Cisplatin

2.5. Concurrent Therapy

3. Summary of the Invention

4. Description of the Invention

4.1. Aminoglycoside-Ligand Preparations

4.1.1. Evidence of Complex Formation

4.2. Polyene and Polyene Macrolide Antibiotic-Ligand Preparations

4.2.1. Evidence of Complex Formation

4.3. Adriamycin-Ligand Preparations

4.4. Cisplatin-Ligand Preparations

5. Example: Streptomycin Preparations

5.1. Reduced Toxicity of Streptomycin-Ligand Preparations

5.2. Antibiotic Activity of Streptomycin-Phosphorylcholine Preparations

6. Example: Reduced Toxicity of Various Aminoglycoside Antibiotic-LigandPreparations

7. Example: Amphotericin B Preparations

7.1. Interaction of Amphotericin B with Water Soluble Cholesterol inVarious Solvents

7.2. Stability of Amphotericin B Preparations

7.3. Reduced Toxicity of Amphotericin B Ligand Preparations

7.3.1. Toxicity of PEG-Cholesterol

7.3.2. Comparison of Toxicity of Amphotericin B and AmphotericinB/PEG-Cholesterol

7.3.3. Toxicity of Various Amphotericin B Preparations in Mice

7.3.4. Reduced Toxicity of Amphotericin B Preparations in Cell Culture

7.3.5. Comparison of Hemolytic Properties of Amphotericin B Preparations

1. FIELD OF THE INVENTION

This invention relates to reducing or "buffering" the toxicity of drugswhose side effects are mediated through binding to endogenous cellulartoxicity receptors, generally lipids. In particular, the drug isadministered in combination with certain ligands such that the toxiceffects of the drug are greatly reduced; however, the drug retainspharmacological activity.

2. BACKGROUND OF THE INVENTION

Several theories have been advanced to explain the mechanism by which avariety of toxic substances exert their desirable therapeutic anddetrimental toxic effects in animals, including humans. Growing evidencesuggests that these substances exert their effects by disturbing thecellular membranes (Schacht et al., 1983, "Aminoglycoside-cell ReceptorInteractions: Implications for Toxicity and In Vitro Models," ThirteenthInternational Congress of Chemotherapy). More particularly, it isbelieved that the drugs bind to certain specific receptors which mediatethe molecular activities responsible for their therapeutic and toxiceffects in animals.

Theories of pharmacological action have been developed for theaminoglycoside antibiotics and for several other drugs, including thepolyene and polyene macrolide antibiotics, a class of antifungal agents,as well as for adriamycin and cisplatin, known antineoplastic agents.These drugs, among others, exert their desirable therapeutic effects bybinding to specific bacterial, fungal or tumor cellular receptors. Theirundesirable toxic effects, on the other hand, are thought to be due tobinding to specific toxicity receptors of non-drug target cells of thetreated animal. Lipids have been suggested as toxicity receptors forthese and other drugs.

Since theories have been more developed for the above-listed drugs,these will be discussed in greater detail below. It is to be understood,however, that drugs other than these may also exert their toxic effectsthrough similar lipid-mediated mechanisms, and that the presentinvention encompasses all of such drugs.

2.1. THE AMINOGLYCOSIDE ANTIBIOTICS

The aminoglycoside antibiotics (e.g., streptomycin, gentamycin,kanamycin, tobramycin, etc.) are used almost exclusively to treatinfections caused by bacteria. Their mode of bactericidal actioninvolves inhibition of protein synthesis in susceptible microorganisms.Some susceptible microorganisms include Escherichia spp., Haemohilusspp., Listeria spp., Pseudomonas spp., Nocardia spp., Yersinia spp.,Klebsiella spp., Enterobacter spp., Salmonella spp., Staphyloccocusspp., Streptococcus spp., Mycobacteria spp., Shigella spp., and Serratiaspp., to name but a few.

The antibiotics in the aminoglycoside group all contain amino sugars inglycocidic linkages. They are polycations and their polarity isprimarily responsible for the pharmacokinetic properties shared by themembers of the group. For instance, these drugs are not adequatelyabsorbed after oral administration, they do not easily penetrate thecerebrospinal fluid, and they are rapidly excreted by the kidney.

Serious toxicity is a major limitation to the usefulness ofaminoglycosides. Three types of toxicity are often encountered with theuse of aminoglycosides: (1) ototoxicity, which can involve both auditoryand vestibular functions of the eighth cranial nerve; (2)nephrotoxicity, which is manifest as acute tubular damage; and (3) acutetoxicity, which can follow intrapleural and intraperitonealadministration and is manifest as a neuromuscular blockade culminatingin respiratory distress.

Ototoxicity involves labyrinthine dysfunction. Nearly 75% of patientsgiven 2 g of streptomycin daily for 60 to 120 days manifest somevestibular disturbance; reduction of the dose to 1 g daily decreases theincidence to 25%. The effects occur in stages: (1) the acute stage (1 to2 weeks) is characterized by nausea, vomiting and equilibratorydifficulty including vertigo; (2) the acute state ends suddenly and isfollowed by chronic labyrinthitis characterized by ataxia; and (3) thechronic stage may persist for two months and is followed by acompensatory stage in which symptoms are latent and appear only when theeyes are closed. Full recovery of coordination may require 12 to 18months, and some patients have permanent residual damage. There is nospecific treatment for the vestibular deficiency.

In order to prevent or reduce the incidence and severity of the toxiceffect on vestibular function, the aminoglycoside dose, the duration oftherapy, and careful observation of the effects on the patient must beconsidered.

The toxic effect of aminoglycosides such as streptomycin and gentamycinis greater upon the vestibular than auditory component of the eighthcranial nerve, however, a decrease in hearing occurs in an appreciablenumber of patients (4-15% of individuals receiving the drug for morethan 1 week) and, in rare cases, complete deafness can occur. Althoughthe location of lesions responsible for vestibular and auditorydysfunction is disputed, it is believed that aminoglycosides destroy theventral cochlear nuclei in the brain stem with extension of pathologicalchanges to the terminals of the nerve fibers in the cochlea.

Nephrotoxicity caused by aminoglycosides is essentially a form of acutetubular necrosis and is initially manifested by the inability toconcentrate urine. The very high concentrations of aminoglycosideantibiotics which accumulate in the renal cortex and urine correlatewith the potential of these drugs to cause nephrotoxicity. For thisreason, neomycin, the most nephrotoxic aminoglycoside, is not generallyadministered systemically in humans. Gentamycin seems to be the mostnephrotoxic of the commonly used drugs.

Typically, after 5 to 7 days of aminoglycoside therapy, kidney damage,manifested by acute tubular necrosis and inability to concentrate urine,occurs and progresses as administration of the drug continues. At thisstage, the urine contains protein and tubular cell casts. A reduction inglomerular filtration rate follows and is associated with elevation inthe concentrations of the aminoglycoside, creatinine, and urea inplasma. Histopathology includes acute tubular damage with secondaryinterstitial damage. These changes are usually reversible andregeneration of renal cells occurs if the drug is discontinued.

Another serious effect of various aminoglycosides is the potentiallyfatal neuromuscular reaction which may develop when a drug such asstreptomycin is instilled into the peritoneal cavity postoperatively (apractice in some surgical clinics). Acute muscular paralysis anddifficulty in respiration may occur due to blockade of the neuromuscularjunction by aminoglycosides. Neuromuscular blockade has also beenobserved in man following intravenous, intramuscular, and oraladministration of these agents. The neuromuscular block may occur byinhibition of acetylcholine release from the preganglionic terminal(through competition with calcium ions) and perhaps to a lesser extentby stabilization of the post junctional membrane. The blockade isantagonized by calcium salts, but only inconsistently byanticholinesterase agents.

These neuromuscular reactions are shared to varying degrees by severalother aminoglycoside antibiotics, particularly neomycin and kanamycin,and to a lesser extent by gentamycin, viomycin, paromomycin andtobramycin. Interestingly, the order of increasing ability of theaminoglycosides to affect acute toxic reactions seems to correlate withtheir ability to affect nephrotoxicity. That the underlying mechanism ofthese toxic reactions may be related is suggested by work associatingthe stimulation or depression of bioelectric events in nerve membraneswith phosphoinositide metabolism (Hokin, 1969, Structure and Function ofNervous Tissue, Bourni, ed., Academic Press, New York, Vol. 3, pp.161-184; Schact and Agranoff, 1972, J. Neurochem. 19:1417-1421; Margolisand Heller, 1966, J. Pharmacol. Expt. Ther. 74:307-312) and similar workassociating phosphoinositide metabolism with aminoglycoside-inducedototoxicity and nephrotoxicity (Orsulakova et al., 1976, J. Neurochem.26:285-290; Schibeci and Schacht, 1977, Biochem. Pharmacol.26:1769-1774; Alexander et al., 1979, J. Antibiotics 32:504-510).Aminoglycoside antibiotics have been shown to bind topolyphosphoinositides in inner ear tissue and kidney in vivo. It hasbeen postulated that phosphatidylinositol bisphosphates(phosphatidylinositol diphosphate) serve as in vivo receptors foraminoglycoside (hereinafter referred to as putative aminoglycosidetoxicity receptors) and render tissues susceptible to these drugs (Lodhiet al., 1980, Biochem. Pharmacol. 29:597-601).

See "The Pharmacological Basis of Therapeutics," 6th edition, Goodmanand Gilman, eds., 1980, Ch. 51, pp. 1162-1199, for a review of theaminoglycosides.

2.2. THE POLYENE AND POLYENE MACROLIDE ANTIBIOTICS

Polyene and macrolide antibiotics are a group of substances obtainedfrom species of actinomycetes. For a review of the pharmacology ofvarious polyene and polyene macrolide antibiotics, see "ThePharmacological Basis of Therapeutics", 6th edition, Goodman and Gilman,eds., 1980, pp. 1233-1236 and Medoff et al., 1983, Ann. Rev. Pharmacol.Toxicol. 23:303-330.

A large number and variety of untoward toxic effects may be associatedwith the use of these drugs. In particular, the toxic effects ofamphotericin B include anaphylaxis, thrombopenia, flushing, generalizedpain, convulsions, chills, fever, phlebitis, headache, anemia, anorexia,and decreased renal function. The toxic effects of the polyene andpolyene macrolide antibiotics appear to result from binding to thetoxicity receptor, cholesterol, a sterol present in animal cells but notfungal cells.

The polyene and polyene macrolide antibiotics appear to exert theirtherapeutic effects against fungi by binding to ergosterol, a sterolpresent in fungal cells but not in human cells. For example,amphotericin B binds in vitro to ergosterol with approximately ten timesgreater affinity than to cholesterol, the putative toxicity receptor.Bittman et al., 1982, Biochim. Biophys. Acta 685:219.

Encapsulating amphotericin B in liposomes has been suggested toattenuate the untoward effects of the drug. Taylor et al. (1982, Ann.Rev. Respir. Dis. 125:610-611) reported that liposome encapsulationsignificantly alters the drug's toxic effects. Their data indicates thatencapsulation probably changes the tissue distribution of the drug. Theyobserved reduced acute toxicity and a maximal tolerable dose nine timesgreater than the maximal tolerable dose for free amphotericin B.Liposome encapsulation of amphotericin B also prolongs the survival ofmice infected with Histoplasma capsulatum. New et al., (1981, J.Antimicrob. Chemother. 8:371-381) have used amphotericin B withcholesterol for the purpose of treating leishmaniasis. Increasedeffectiveness of the drug was observed.

Sodium deoxycholate is used in a commercial preparation, Fungizone, toeffect a colloidal dispersion of amphotericin B, buffers and diluents.Fungizone is available packaged in vials as a sterile, lyophilizedpowder to which water is added. This mixture is shaken and added to a 5%dextrose solution. The resulting solution can then be administered as aninjection.

2.3. ADRIAMYCIN

Adriamycin, an antineoplastic drug, is a glycosidic anthracyclineantibiotic that is a fermentation product of the fungus Streptomycespeucetius var. caesius. Adriamycin has a tetracycline ring structurewith an unusual sugar, daunosamine, attached by a glycosidic linkage.

Adriamycin is effective in the treatment of acute leukemias andmalignant lymphomas and in a number of solid tumors. The drug isparticularly beneficial in a wide range of sarcomas, includingosteogenic, Ewing's, and soft-tissue sarcomas. It is one of the mostactive single agents for the treatment of metastatic adenocarcinoma ofthe breast, carcinoma of the bladder, bronchogenic carcinoma andneuroblastoma.

Myelosuppression is a major dose-limiting complication with adriamycin.Stomatitis, gastrointestinal disturbances, and alopecia are common butreversible. Cardiomyopathy is a unique characteristic of theanthracycline antibiotics. Tachycardia, arrhythmias, and ST-T wavechanges in the ECG may be early manifestations of cardiac toxicity.Severe and rapidly progressive congestive heart failure may follow.Nonspecific alterations, including a decrease in the number ofmyocardial fibrils, mitochondrial changes, and cellular degeneration,are visible by electron microscopy. In view of this cardiotoxicty,therapeutic utility of adriamycin is limited.

Because adriamycin is primarily metabolized and excreted by the liver,it is important to reduce the dosage in patients with impaired hepaticfuntion.

The antitumor effects of adriamycin appear to result from intercalationof the molecule between adjacent base pairs of DNA, thus interferingwith the proliferation of rapidly dividing tumor cells. Toxic effectsappear to be mediated by binding of the drug to cardiolipin, theputative toxicity receptor. Goormaghtigh et al., have shown thatadriamycin adsorbs in vitro to cardiolipin and, to lesser degrees, tophosphatidylserine and phosphatidic acid (1980, Biochem. Pharmacol.29:3003-3010). There appears to be a relatively strong electrostaticinteraction between protonated amine groups of the sugar residues ofadriamycin and ionized phosphate residues of the cardiolipin,concomitant with interaction between adjacent anthraquinonechromophores. The association constant for cardiolipin/adriamycin isabout 1.6×10⁶ mol⁻¹, while the association constant for DNA/adriamycinis about 2.4×10⁶ mol⁻¹. Thus, adriamycin has a higher affinity for DNAthan for cardiolipin.

Fluorescent studies have revealed that adriamycin binds to the headgroup of cardiolipin. Goormaghtigh et al., 1980, Biochim. Biophys. Acta597:1-14.

Goormaghtigh et al., also observed a good correlation between binding ofadriamycin derivatives to cardiolipin and mitochondrial toxicity (1980,Biochem. Pharmacol. 29:3003-3010). Thus, it has been proposed thatcardiolipin is the toxicity receptor for adriamycin in animals(Goormaghtigh et al., 1982, Biochem. Biophys. Res. Comm. 104:314-320).

It has been proposed to encapsulate adriamycin in liposomes (Forssen andTokes, 1983, Cancer Res. 43:546-550). Liposome encapsulation changes thetissue distribution of adriamycin; the free drug appears predominantlyin the liver and spleen. There is an apparent reduction in chroniccardiotoxicity and an increase in antileukemic activity.

2.4. CLISPLATIN

Cisplatin is an inorganic, water soluble, platinum containingcoordination complex (planar), containing ammonium and chloride residuesin the cis configuration. Despite pronounced nephrotoxicity andototoxicity, cisplatin is very useful in combination chemotherapy ofmetastatic, testicular and ovarian carcinoma. Encouraging effects havealso been reported during treatment of tumors of the bladder and of thehead and neck.

For a review of the pharmacology of cisplatin, see "The PharmacologicalBasis of Therapeutics", 6th edition, Goodman and Gilman, eds., 1980, pp.1298-1299.

Studies on crystal structure analysis of cisplatin have demonstrated astrong binding of these compounds to guanosine and related inosinederivatives (e.g., inosine 5'-monophosphate) (Louie and Bau, 1977, J.Amer. Chem. Soc. 99:3874-3876; Goodgame et al., 1975, Biochim. Biophys.Acta 378:153; Bau et al., 1977, J. Clin. Hematol. Oncol., Wadley MedicalBulletin 7:51). Thus, the mechanism of action leading to thenephrotoxicity and ototoxicity observed upon administration of cisplatinmay be similar to that for the aminoglycoside antibiotics (see Section2.1.) and ligands useful for buffering aminoglycosides may be used forbuffering cisplatin.

2.5. CONCURRENT THERAPY

Combinations of antimicrobial agents have been widely described for thetreatment of infections. In fact, combination therapy using a number ofdifferent drugs is recommended for the treatment of many cancers.

Concurrent therapy, however, with certain pharmaceutical agents may becomplicated because agents which exert a synergistic effect in vitrocannot be formulated in a single mixture to use in vitro. Mixtures ofgentamycin and nafcillin, a penicillin, at therapeutically effectiveconcentrations result in the formation of complexes that precipitate outof solution and, therefore, cannot be administered in vivo together. Infact, certain drug combinations are not recommended for use in vivo dueto drug incompatibility (i.e., either inactivation of the drug orformation of a precipitate). For example, it is recommended that thefollowing antibiotics not be mixed with any other drug: gentamycin,kanamycin, lincomycin, cephalothin, and ampicillin (Davis and Abbitt,1977, JAVMA 170(2):204-207). Moreover, certain agents cannot besolubilized in the same media due to chemical restraints (e.g., a lipidsoluble compound and a water soluble compound).

3. SUMMARY OF THE INVENTION

The present invention relates to preparations of certain drugs inadmixture with various ligands that reduce the toxicity of the drugs invivo. When these drugs are administered in combination with the ligandsdescribed herein, toxicity of the drug is greatly reduced while the drugretains pharmacological activity. Drugs whose toxic effects may bereduced or "buffered" by the method of the invention include those inwhich toxic side effects are mediated through binding to endogenouscellular substrates, which are generally lipids. The term "ligand" asused herein includes compounds which reduce the toxicity of the drugs inthe preparation; the term is not meant to indicate the type ofinteraction or reaction between such compounds and the drug.

A particularly useful advantage of the drug-ligand preparationsdescribed herein is that high doses of drug may be administered to ananimal or human with lessened deleterious toxic effects than observedwith conventional drug preparations. Thus, maintenance doses may begiven less often to achieve acceptable serum levels. Since higher dosesof drug may be administered without the usual toxic effects, thepreparations of the invention allow for a greater margin of error inselection of initial doses of drug.

The preparations of the invention can be viewed as an entirely new classof therapeutic compositions possessing all of the advantages of theconventional drug preparations with reduction of the major clinicallyrelevant disadvantages.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention involves drug preparations of reduced toxicity. Adrug-ligand preparation is made such that, when administered to ananimal or human, the toxicity of the drug in the preparation is reducedor eliminated, yet the drug retains pharmacological activity.

The ligand to be used in the preparations of the invention depends onthe particular drug used and the relevant toxicity receptor. Generally,the drugs which may be used in practicing the present invention arethose whose in vivo toxicity to mammalian cells is mediated throughbinding to lipid toxicity receptors. Such drugs include monoaminated orpolyaminated drugs, antimicrobials, antivirals, antifungals,antibacterials, aminoglycosides, polyene and polyene macrolideantibiotics, lincosamides, and polymixins, to name a few.

According to the invention, appropriate ligands (lipids or lipid headgroups) to which the drug binds are admixed with the drug prior toadministration. It is believed the ligand binds to the toxicity receptorto prevent interaction between the drug and the receptor. Thiscombination of drug with ligand prevents or reduces the binding of thedrug to its endogenous toxicity receptor, thus reducing or eliminatingthe toxicity of the drug. The drug retains its pharmacologicaleffectiveness.

4.1. AMINOGLYCOSIDE-LIGAND PREPARATIONS

As discussed in Section 2.1., it is believed that phosphatidylinositolbisphosphates serve as in vivo toxicity receptors for the aminoglycosideantibiotics. According to the invention, aminoglycosides are mixed withligands which prevent or reduce the binding of aminoglycosides to theendogenous toxicity receptors. Ligands useful in the aminoglycosidepreparations of the invention include, but are not limited to, certainphosphate esters, phosphate anhydrides and sulfatides.

The ligands which may be used in the present invention are not equallyeffective in reducing aminoglycoside toxicity (see Section 5.1., infra).The ability of these ligands to reduce toxicity of aminoglycosideantibiotics appears to correlate with two phenomena. The firstphenomenon is the degree to which various ligands appear to associatewith the aminoglycosides by virtue of the strength of dipole-dipoleinteraction and subsequent hydrogen bonding. Ligands exhibitingextensive resonance structures, whereby electron delocalization iseasily visualized, seem to provide the most protection againstaminoglycoside toxicity. Ligands exhibiting lower potential for hydrogenbonding provide only intermediate protection. The configuration ofnegative charges on phosphatidylinositol bisphosphate (the putativeaminoglycoside toxicity receptor) allow for a specific, three pointbinding to positively charged aminoglycosides. Although a strongcorrelation exists between the charge of aminoglycosides and theirrelative toxicity (Schact, 1983, Proceedings of the ThirteenthInternational Congress of Chemotherapy), some aminoglycosides do not fitthis pattern of charge versus toxicity and, thus may be stabilized intheir binding to phosphatidylinositol bisphosphate by hydrogen bonding.Thus, the ligands that most effectively reduce the toxicity seem to havethe same binding characteristics of phosphatidylinositol bisphosphate.

The second phenomenon concerning the efficacy of various ligands toreduce aminoglycoside toxicity concerns a correlation of the ability ofthese ligands to bind C-reactive protein. C-reactive protein is an acutephase protein appearing in the serum of man during various pathologicconditions. It is precipitated from such serum by pneumococcalC-polysaccharide in the presence of calcium ions. Phosphorylcholine andlysophosphatidylcholine bind strongly to C-reactive protein, whilephosphorylethanolamine and glycerolphosphorylcholine bind less strongly(Volanakis and Kaplan, 1970, Proc. Soc. Exp. Biol. Med. 136:612-614).Coincidentally, phosphorylcholine and lysophosphatidylcholine are veryeffective in reducing aminoglycoside toxicity whereasphosphorylethanolamine and glycerolphosphorylcholine are less effective.The correlation of the ability of these ligands to bind C-reactiveprotein and the ability to reduce aminoglycoside toxicity whileretaining antimicrobial activity remains unexplained. This correlationis useful as a predictive tool to select candidates for ligands usefulin the preparations of the invention, but it is to be understood thatthis does not necessarily indicate the mechanism of action by which thepreparations of the invention exert the observed effects.

The ligands which are used in the present invention to attenuateaminoglycoside toxicity include, but are not limited to:lysophosphatidylcholine, phosphorylcholine, tripolyphosphate,phosphorylserine, phosphoglyceric acid, inositol monophosphate, inositolbiphosphate, inositol triphosphate, inositol tetraphosphate, inositolpentaphosphate, inositol hexaphosphate, and phosphorylinositols; i.e.,phosphate esters and phosphate anhydrides. In addition, lipoteichoicacids, teichoic acids (including pneumococcal and streptococcalC-polysaccharide), sulfatides, nucleotides, stearylamines,diacylglycerol, aminocaproic acid, pyridoxal phosphate, chondroitonsulfate, cysteic acid, p-aminophenyl phosphate, p-aminophenyl sulfate,poly-1-lysine, poly-1-arginine, poly-1-glutamic acid, poly-1-asparticacid and other polymeric and monomeric amino acids will efficaciouslyreduce toxicity.

Further, phospholipids such as cardiolipin,phosphatidylinositolphosphates, including phosphatidylinositol phosphateand phosphatidylinositol diphosphate, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, galactocerebroside sulfate,phosphatidylinositol and phosphatidic acid, all of which willspontaneously form liposomes at excess concentrations in water (abovetheir critical micelle concentrations) would also be expected to reduceaminoglycoside toxicity. Such aminoglycoside-containing liposomes wouldallow aminoglycosides to be given safely above the LD₁₀ or LD₅₀ of thefree drug. For example, the LD₅₀ of streptomycin in mice is 741 mg/kg(see Table IV infra); encapsulation of streptomycin in liposomes wouldallow the administration of this concentration of streptomycin withoutobserving any acute toxicity.

In a particular embodiment, the phospholipids phosphatidylinositolphosphate and phosphatidylinositol bisphosphate may be especially usefulwhen used at concentrations greater than their critical micelleconcentrations; that is, with the aminoglycoside antibiotic encapsulatedwithin liposomes of these phospholipids. As discussed above, thesephospholipids are the putative toxicity receptors for aminoglycosides,and for this reason, they are especially desirable for use as ligands inthe preparations of the invention, either above or below their criticalmicelle concentrations (i.e., in liposomes or in solution).

The preparations of the invention may be used in situations requiringhigh initial concentrations and/or high levels of sustained antibiotictherapy. They may also be useful in renal compromised patients in whichinitial toxicity to the patient is a concern. The invention also enablesadministration of antibiotics at levels higher than currently acceptablelevels for sustained minimal inhibitory serum concentrations. By virtueof the ability to administer higher loading dosages of antibiotics,higher sustained levels of antibiotics are possible. The technology ofthe invention may also be applied in veterinary therapy to situationsincluding, but not limited to, gram-negative enteritis, mastitis,"shipping fever", infectious keratoconjunctivitis, vibrosis,mycobacterial infections and Brucella spp. infections.

The aminoglycoside antibiotics which may be used in the presentinvention include but are not limited to: streptomycin, gentamycin,tobramycin, amikacin, kanamycin and neomycin.

According to the present invention, an aminoglycoside antibiotic ismixed with a ligand prior to administration to an organism in vivo.Sterile water is the preferred diluent. Due to the nature of theaminoglycoside antibiotics, parenteral administration of the preparationis the preferred route. The aminoglycoside and ligand may be mixedimmediately prior to administration in vivo. Alternatively, theaminoglycoside and ligand may be mixed for 1 hour, 2 hours, or longerprior to administration, depending on the stability of theaminoglycoside. Using either method, toxicity is reduced and theantibiotic retains antimicrobial activity. As a result, increased dosesof antibiotic may be administered to animals or humans with greatersafety.

The reasons for the attenuation of toxicity remain obscure, and a numberof mechanisms seem plausible. Possibly the ligands form a complex withthe aminoglycosides, thus preventing the binding of the antibiotic toits putative aminoglycoside toxicity receptor in vivo. It is alsoconceivable that association between aminoglycosides and ligands occursby virtue of dipole interactions, resulting in an array of hydrogenbonds between them. At physiological pH, the guanidino groups ofaminoglycosides are fully protonated and are stabilized by resonance.These groups are particularly good sites for hydrogen bond interactionswith phosphate groups (examples of which are the binding of NAD⁺ toarginine 101 of lactate dehydrogenase or the binding of thymidine 3'-5'diphosphate to the guanidinian ions of arginines 35 and 87 ofstaphylococcal nuclease). Guanidino groups are capable of forming up tofive hydrogen bonds each, and can interact simultaneously with phosphategroups and adjacent groups such as carbonyls. Alternatively, the ligandsmay bind to the putative aminoglycoside toxicity receptors themselves,thus preventing the binding of the antibiotic.

4.1.1. EVIDENCE FOR COMPLEX FORMATION

The following observations and data suggest that anaminoglycoside-ligand complex is formed which is less toxic than theaminoglycoside alone:

(1) SDS-Polyacrylamide Gel Electrophoresis: Equal volumes of mouse serumwere incubated for 0.5 hour and 2 hours with ¹²⁵ I-gentamycin sulfate(¹²⁵ I-GS) or with ¹²⁵ I-gentamycin-phosphorylcholine (¹²⁵ I-GPC), 1:3molar ratio, at 1×10⁵ cpm per sample. Aliquots of the incubated serumsamples and aliquots of ¹²⁵ I-GS as well as ¹²⁵ I-GPC were then preparedfor sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)by boiling the samples in β-mercaptoethanol (β-ME), SDS, and urea for 1to 2 minutes as described by Laemmali (1970, Nature 227:680). Afterelectrophoresis the gel was stained with Coomassie-Blue (C-Blue) andexposed to X-ray film (autoradiographed) at -70° C. In the lanerepresenting the serum sample incubated with ¹²⁵ I-GS and in the lanerepresenting the serum sample incubated with ¹²⁵ I-GPC all serumpolypeptides were stained with C-Blue but none of the serum polypeptidebands were radioactively labeled. However, in the lane representing theserum sample sample incubated with ¹²⁵ I-GPC a low molecular weight band(i.e., lower than the molecular weights of the serum polypeptides)stained intensely with C-Blue; this band was also radioactive asdetermined by autoradiography. This low molecular weight band was notpresent in the lane representing serum incubated with ¹²⁵ I-GS. In thelane representing ¹²⁵ I-GS (without sera) no bands were detected byautoradiography, but in the lane representing ¹²⁵ I-GPC (without sera)the same low molecular weight, radioactively-labeled band which stainedwith C-Blue was detected.

Since phosphorylcholine stains intensely with C-Blue, this low molecularweight band which is stained by C-Blue and is also radioactively labeledrepresents the putative ¹²⁵ I-gentamycin-phosphorylcholine complex.Interestingly, the putative complex did not dissociate under thedenaturing conditions used in the sample preparation for SDS-PAGE.

(2) Dissociation of the Putative Complex: If the aminoglycoside-ligandmixture is prepared or administered in an appropriate "high-salt"solution (a high salt concentration has been shown to inhibit formationof complexes or cause dissociation of some complexes), the toxicity ofthe aminoglycoside is not reduced. One explanation for this observationis that dispersing the aminoglycoside-ligand mixture in 0.6M ammoniumacetate dissociates the complex formed between the aminoglycoside andthe ligand. In support of this, see Schact, 1978, J. Lipid Res.19:1603-1607, where 0.6M ammonium acetate is reported to elutephosphatidylinositol phosphate which was bound to neomycin immobilizedon a column. When an aminoglycoside-ligand preparation dispersed in 0.6Mammonium acetate is administered subcutaneously to mice, it appears thattoxicity is not significantly reduced (see Table I).

(3) Separate Administration of Ligand and Aminoglycoside: If the ligandis administered to an animal before the the aminoglycoside isadministered, the toxicity of the aminoglycoside is not reduced to thesame extent that a mixture of streptomycin and phosphorylcholine reducestoxicity (see Table I).

                  TABLE I                                                         ______________________________________                                        COMPARISON OF TOXICITY OF STREPTOMYCIN                                        ADMINISTERED WITH AND WITHOUT LIGAND                                                            Lethality                                                                     No. Dead/No.                                                                              Percent                                         Treatment.sup.1   Inoculated  Lethality                                       ______________________________________                                        Control           7/8         87.5                                            Streptomycin                                                                  Aminoglycoside + Ligand.sup.2                                                                   1/8, 0/6    7                                               (Streptomycin +                                                               Phosphorylcholine)                                                            Aminoglycoside + Ligand                                                                         3/6         50                                              in Ammonium Acetate Buffer.sup.3                                              (Streptomycin +                                                               Phosphorylcholine)                                                            Aminoglycoside Administered                                                   After Inoculation of Ligand.sup.4                                             (Min. post-inoculation)                                                        0                5/8         62.5                                            10                5/8         62.5                                            30                6/7         85.7                                            ______________________________________                                         .sup.1 In all trials the streptomycin dose was 1000 mg/kg body weight         inoculated subcutaneously in Swiss Webster mice. In all trials where          ligand was administered, the molar ratio of aminoglycoside to ligand was      1:3.                                                                          .sup.2 The aminoglycoside + ligand mixture was prepared by mixing             streptomycin and phosphorylcholine immediately prior to administration.       .sup.3 The streptomycin + phosphorylcholine mixture was prepared in 0.6M      ammonium acetate. Subcutaneous injection of ammonium acetate alone (0.6M)     was not lethal (0/6 lethality).                                               .sup.4 In this trial both the phosphorylcholine and streptomycin were         inoculated subcutaneously at different sites after the time spans             indicated.                                                               

It appears that reduction in aminoglycoside toxicity is related to theformation of a complex between the aminoglycoside and ligand for thefollowing reasons: (1) drug toxicity is not significantly reduced whenthe aminoglycoside and ligand are administered in ammonium acetate (atconcentrations which are known to dissociate or prevent formation ofsome complexes); and (2) separate administrations of the ligand followedby drug were not effective in reducing toxicity of the streptomycin.However, the invention is not to be limited to this theory ofinteraction between the aminoglycoside and ligand. Other potentialexplanations for the observed reduction in toxicity are discussed above.

4.2. POLYENE AND POLYENE MACROLIDE ANTIBIOTIC-LIGAND PREPARATIONS

As described in Section 2.2., the polyene and polyene macrolideantibiotics are believed to exert their toxic effects in humans andanimals by binding to cholesterol, the putative toxicity receptor. Thus,ligands suitable for reducing the toxicity in mammals of the polyene andpolyene macrolide antibiotics, such as amphotericin B, are those whichprevent binding to endogenous cholesterol. Such ligands would includesterols and water soluble derivatives thereof, and, in particular,cholesterol and its water soluble derivatives such as polyethyleneglycol-cholesterol (PEG-cholesterol), cholesterol hemisuccinate (CHS),and the like. The water soluble cholesterol derivatives are ofparticular utility since clear solutions are highly preferred forinjection.

The lipid ligands of this particular embodiment of the present inventionmay be prepared at a concentration below that used for micelle formationand are thus free of liposomes. Since liposomes are not present, thereis no difficulty with respect to stability of the preparations.Furthermore, administration of cholesterol-containing liposomes couldresult in the deposition of the liposomes primarily in the liver andspleen, which may be undesirable. Additionally, one need not beconcerned about administering a particulate preparation which may clogor reduce circulation. Thus, effective preparations of polyene orpolyene macrolide antibiotics mixed with water soluble cholesterol or acholesterol derivative, yet free of liposomes, are highly effective inreducing toxicity of the drugs.

Amphotericin B, a polyene antibiotic, is not soluble in water and, forinjections, is commercially solubilized in sodium deoxycholate whichalso has toxic properties (Fungizone, E. R. Squibb & Sons, Princeton,N.J.). Consequently, because of the two toxic components of thecommercial pharmaceutical agent, therapeutic doses of Fungizone oramphotericin B-deoxycholate (amphotericin B-DOC) must be kept low. Aswill be seen in the examples following, both a reduction in toxicity andsolubilization of Amphotericin B can be obtained by solubilizing theamphotericin B in a water soluble cholesterol preparation. Reduction intoxicity of an amphotericin B administration is obtained in two ways:

(1) Introduction into the system of toxic deoxycholate is eliminatedsince non-toxic water soluble cholesterol is used as the solubilizer;and

(2) Water soluble cholesterol, when added to amphotericin B, blocksaffinity for in situ lipid binding sites on the drug, thus blocking thetoxicity pathway.

4.2.1. EVIDENCE FOR COMPLEX FORMATION

As indicated in Section 4.1., the reasons for the attenuation oftoxicity seen when polyene or polyene macrolide antibiotics areadministered in combination with the ligands of the present inventionremain obscure. One possbility is that the ligand forms a complex withthe polyene antibiotic thus preventing binding of the drug to itsputative toxicity receptor.

The following observations and data suggest that such a complex isformed between amphotericin B and the ligand PEG-cholesterol.

Spectrophotometric Analysis: The absorbance spectrum of amphotericin Bin water demonstrates three peaks at the following wavelengths: 386,407, 418 nm. When Fungizone is mixed with PEG-cholesterol in an aqueoussolution, the peak at 407 nm disappears. The absence of an absorptionpeak at about 407 nm is evidence of the amphotericin B-cholesterolcomplex formation. However, the absorbance spectra of Fungizone inmethanol and Fungizone mixed with PEG-cholesterol in methanol areidentical. This indicates that no complex is formed in methanol (SeeSection 7 for details).

4.3. ADRIAMYCIN-LIGAND PREPARATIONS

The cardiotoxicity of adriamycin appears to be mediated by binding ofthe drug to cardiolipin or cardiolipin head groups (see Section 2.3.).Accordingly, these preparations of the present invention utilizeadriamycin in admixture with ligands which block the binding ofadriamycin to endogenous cardiolipin. Such ligands would includecardiolipin or cardiolipin head groups.

As with the polyene and polyene macrolide-ligand preparations of Section4.2., the preparations of the invention do not contain liposomes. Hence,none of the disadvantages of liposome administration are encounteredwith the preparations of the invention.

4.4. CISPLATIN-LIGAND PREPARATIONS

The nephrotoxicity and ototoxicity of cisplatin are apparently mediatedin a similar fashion to that of the aminoglycoside antibiotics (seeSection 2.4.). Consequently, any ligand which interferes with thebinding of cisplatin to phosphatidylinositol bisphosphate is suitablefor buffering toxicity of cisplatin.

5. EXAMPLES: STREPTOMYCIN PREPARATIONS

The data presented in the following examples demonstrate the reductionof toxicity of aminoglycoside antiobiotics which retain antimicrobialactivity when administered as an aminoglycoside-ligand preparation.

5.1. REDUCED TOXICITY OF STREPTOMYCIN-LIGAND PREPARATIONS

The following data (presented in Table II) compare the acute toxicity ofstreptomycin administered subcutaneously in mice to that of streptomycinadministered in combination with various ligands.

For each trial 200 mg of streptomycin sulfate and one of the followingligands was dissolved in distilled water (final volume 5 ml) to obtain a1:3 molar ratio or a 1:1 molar ratio of streptomycin to ligand: 260 mgegg lysophosphatidylcholine (1:3); 87 mg egg lysophosphatidylcholine(1:1); 129 mg phosphorylcholine (1:3); 110 mg inositol hexaphosphate(1:1); 80 mg tripolyphosphate (1:1); 71 mg phosphorylethanolamine (1:3);70 mg choline chloride (1:3); 93 mg phosphorylserine (1:3); 196 mginosine 5'-monophosphate (1:3); 115 mg phosphoglyceric acid (1:3); or76.5 mg inositol monophosphate (1:1). These solutions were mixed for atleast one hour. Once dissolved, a 0.6 ml aliquot of the solution wasadministered subcutaneously into mice (average weight of 24 gm each) inorder to deliver a dose of 1000 mg streptomycin per kg body weight.

The results in Table II clearly demonstrate that egglysophosphatidylcholine, phosphorylcholine and inositol hexaphosphateare very effective in reducing the toxicity of streptomycin. Theremaining ligands included in Table II vary in their effectiveness inreducing aminoglycoside toxicity.

                                      TABLE II                                    __________________________________________________________________________    COMPARISON OF THE ACUTE TOXICITY OF                                           STREPTOMYCIN ADMINISTERED                                                     WITH AND WITHOUT VARIOUS LIGANDS.sup.1                                                       Molar Ratio                                                                             Lethality                                                           Streptomycin:                                                                           No. Dead/                                                                             Percent                                                     Ligand    No. Inoculated                                                                        Lethality                                    __________________________________________________________________________    Control                  6/8, 5/6, 6/8                                                                         77.7                                         Streptomycin                                                                  Ligand.sup.2                                                                  Egg Lysophosphatidylchloine                                                                  (1:3)     0/8, 0/4                                                                              0                                            Egg Lysophosphatidylcholine                                                                  (1:1)     0/7     0                                            Phosphorylcholine                                                                            (1:3)     0/8, 0/8                                                                              0                                            Inositol hexaphosphate                                                                       (1:1)     0/8     0                                            Phosphoglyceric acid                                                                         (1:3)     3/8     37.5                                         Tripolyphosphate                                                                             (1:1)     3/8, 4/7                                                                              47.3                                         Phosphorylserine                                                                             (1:3)     4/8     50                                           Inositol monophosphate                                                                       (1:1)     5/8     62.5                                         Phosphorylethanolamine.sup.3                                                                 (1:3)     4/7     66                                           Inosine 5'-monophosphate                                                                     (1:3)     7/8     87.5                                         Choline chloride.sup.4                                                                       (1:3)     7/7     100                                          __________________________________________________________________________     .sup.1 In all trials the streptomycin dose was 1000 mg/kg body weight,        inoculated subcutaneously in Swiss Webster mice.                              .sup.2 The following compounds, when mixed with the aminoglycoside,           precipitated out of solution and could not be administered to mice: egg       phosphatidic acid and inositol hexasulfate.                                   .sup.3 Phosphorylethanolamine administered without streptomycin was not       lethal (0/7 lethality).                                                       .sup.4 Choline chloride administered without streptomycin was not lethal      (0/8 lethality).                                                         

5.2. ANTIBIOTIC ACTIVITY OF STREPTOMYCINPHOSPHORYLCHOLINE PREPARATIONS

The following experiment compares the antibiotic activity of sera frommice inoculated subcutaneously with streptomycin to that of sera frommice inoculated with a streptomycin-phosphorylcholine preparation. Theresults demonstrate that the toxicity of streptomycin can be reduced(See Table II) while retaining antimicrobial activity (See Table III).

Adult Swiss Webster mice were inoculated subcutaneously with 0.6 ml ofeither streptomycin at a concentration of 18.3 mg/ml (400 mg/kg bodyweight) or a phosphorylcholine-streptomycin preparation (antibiotic asabove and phosphorylcholine 11.5 mg/ml, i.e., 255 mg/kg body weight). At2, 4, 6, 8.5 and 19 hours post-inoculation, three mice from each groupwere anesthetized with ether and the retro-orbital blood was collectedindividually using heparinized capillary pipets. After the lastbleeding, the blood was centrifuged to separate serum from cells. Theantibiotic activity of the sera was determined in 96-well, U-shapedmicroplates as follows.

Each serum sample (50 μl) was mixed with 50 μl tryptosephosphate broth.These were serially diluted in the microplate wells (50 μl aliquots werediluted into 50 μl of broth serially). Then, 50 μl of broth containing10³ colony-forming units of Staphylococcus aureus (ATCC No. 14154) wereadded to each well and the microplates were incubated overnight at 37°C. The endpoint of the titration was indicated by that dilution of serumin which the growth of S. aureus in micro-well culture was inhibitedapproximately 50% as compared to the control cultures which received noantibiotics.

The results (Table III) indicate that antimicrobial activity of mousesera obtained at different time intervals post-inoculation withantibiotic is similar regardless of whether streptomycin wasadministered alone or in combination with phosphorylcholine. Therefore,the antibiotic retains antimicrobial activity.

                  TABLE III                                                       ______________________________________                                        ANTIBACTERIAL ACTIVITY OF SERA FROM                                           MICE INOCULATED SUBCUTANEOUSLY WITH                                           STREPTOMYCIN-PHOSPHORYLCHOLINE                                                PREPARATIONS                                                                            Titer.sup.1 in Individual Sera                                                of Mice Inoculated with:                                            Hours Post  Streptomycin +                                                    Inoculation Phosphorylcholine                                                                           Streptomycin                                        ______________________________________                                        2           2, 2, 2       2, 2, 2                                             4           4, 4, 4       4, 4, 4                                             6           8, 8, 8       8, 8, 8-16                                          8.5         2-4, 2-4, 2   2, 2, 2                                             19          2, 2, 2       0, 0, 2                                             ______________________________________                                         .sup.1 Reciprocal of the endpoint dilution in the microassay described in     text.                                                                    

6. EXAMPLE: REDUCED TOXICITY OF VARIOUS AMINOGLYCOSIDEANTIBIOTIC-PHOSPHORYLCHOLINE PREPARATIONS

The following data compares the LD₅₀ of various aminoglycosideantibiotics administered with and without ligand.

In order to determined the LD₅₀ of the aminoglycoside preparations aquantity of antibiotic was dissolved in 20 ml distilled water with orwithout phosphorylcholine to achieve a final 1:3 molar ratio ofantibiotic to ligand. Then, 0.6 ml aliquots of serial dilutions of theaminoglycoside preparations were injected subcutaneously into SwissWebster mice (ten animals per group, average weight 23 g each). After 24hours the number of survivors in each group was determined and the LD₅₀and 95% confidence limits for each were computed according to theSpearman-Karber Method for Quantal Data (from Finney, 1952, StatisticalMethod in Biological Assay, Hafner Pub. Co., New York). Results areshown in Table IV.

The results in Table IV clearly demonstrate that the LD₅₀ of eachantibiotic administered in conjunction with ligand is greater than theLD₅₀ of the same antibiotic administered alone.

                  TABLE IV                                                        ______________________________________                                        LD.sub.50 OF AMINOGLYCOSIDE                                                   ANTIBIOTICS ADMINISTERED WITH                                                 AND WITHOUT PHOSPHORYLCHOLINE                                                                          95%                                                                           Confidence                                           Aminoglycoside.sup.1                                                                        LD.sub.50.sup.2                                                                          Interval.sup.3                                                                          LD.sub.50.sup.4                            Preparation   (mg/kg)    (mg/kg)   Ratio                                      ______________________________________                                        Streptomycin  741        616-891   --                                         Streptomycin +                                                                              2123       1725-2570 1:2.9                                      Phosphorylcholine                                                             (1:3)                                                                         Gentamycin    692        602-794   --                                         Gentamycin +  1202        959-1506 1:1.7                                      Phosphorylcholine                                                             (1:3)                                                                         Neomycin      371        301-457   --                                         Neomycin +    912         758-1096 1:2.5                                      Phosphorylcholine                                                             (1:3)                                                                         ______________________________________                                         .sup.1 See text for explanation of subcutaneous administration of             aminoglycoside preparations in Swiss Webster mice.                            .sup.2 The LD.sub.50 is expressed as mg aminoglycoside antibiotic per kg      body weight.                                                                  .sup.3 Ten animals were inoculated in each group.                             .sup.4 The LD.sub.50 ratio is the ratio of the LD.sub.50 of the               aminoglycoside antibiotic to the LD.sub.50 of the aminoglycosideligand        preparation.                                                             

7. EXAMPLES: AMPHOTERICIN B PREPARATIONS

The following subsections described the preparation and the formation ofa complex (as evidenced by spectrophotometric data) between amphotericinB (AmB) a polyene macrolide antibiotic and PEG-cholesterol. The complexappears to remain stable in vivo. Furthermore, the complex has a reducedtoxic effect on cells in vitro and when used in vivo.

7.1. INTERACTION OF AMPHOTERICIN B WITH PEG-CHOLESTEROL IN VARIOUSSOLVENTS

Because amphotericin B is insoluble in aqueous solutions,dimethysulfoxide (DMSO) is used to solubilize the amphotericin B. Theaddition of PEG-cholesterol to the solubilized amphotericin B results inthe formation of a complex between the amphotericin B and thePEG-cholesterol. However, DMSO, which is not approved for internal usein humans, cannot be removed by rotoevaporation. Therefore an alternatemeans of removal must be used. When methanol is added to thepreparation, the methanol forms an easily removable azeotrope with DMSO.When PEG-cholesterol is added concurrently with the methanol, maximumcomplex formation is achieved.

Preparation 1: 25 mg of amphotericin B (AmB) was dissolved in 2.0 ml ofDMSO. Then 30 mg PEG-cholesterol and 23 ml methanol were added to thedissolved amphotericin B. With time a precipitate formed which wasremoved by centrifugation at 10,000×g for 10 minutes. The resultingsupernatant was rotoevaporated to dryness at 55° C.-60° C. The remainingfilm was suspended in 10 ml distilled water and sonicated to clearness.This preparation was designated AmB (25)/PEG-Chol.

Preparation 2: Preparation 2 was prepared identically as for Preparation1 except that 150 mg PEG-cholesterol was used. The preparation wasdesignated AmB(150)/PEG-Chol-2.

Absorbance spectra at a wavelength of 350-550 nm were obtained for thefollowing solutions: (1) Fungizone (a commercially available preparationconsisting of a lyophilized powder of amphotericin B and sodiumdeoxycholate) in either methanol or water; and (2) AmB(25)/PEG-Chol andAmB(150)/PEG-Chol in either water or methanol. The spectrum obtained forFungizone in water consisted of peaks at about 386, 407 and 418 nm. Thespectra obtained for both AmB/PEG-Chol preparations in water consistedof peaks at about 387 and 417 nm; the absence of an absorption peak atabout 407 nm in the spectrum is evidence of an interaction or complexformation between Amphotericin B and PEG-cholesterol. This is furthersupported by the fact that the spectra obtained for Fungizone inmethanol and AmB/PEG-Chol in methanol are identical. The identicalspectra indicates that, in methanol, amphotericin B does not form acomplex with PEG-cholesterol.

7.2. STABILITY OF AMPHOTERICIN B/PEG CHOLESTEROL

A sample of 5 mg of AmB was dissolved in 0.5 ml of DMSO. To this wasadded 10 mg of PEG-cholesterol and 3.0 ml methanol. This solution wascentrifuged at 12,100×g for 10 minutes to remove a small amount offlocculate, rotoevaporated to dryness, resuspended in 10 ml distilledwater adjusted to approximately pH 7.0, and sonicated to clearness. Theresulting clear solution containing the AmB/PEG-cholesterol was dividedinto two equal aliquots (A and B).

The following experiment indicates that the removal of PEG-cholesterolresults in a loss of stability of the complex: CHCl₃ was added dropwiseto aliquot A. The CHCl₃ acted to dissolve or extract the PEG-cholesterolresulting in a precipitation of amphotericin B in the aqueous phase.DMSO, when added dropwise to the precipitate, dissolved the amphotericinB in the aqueous phase.

In order to use the AmB/PEG-cholesterol complexes in vivo, it isimportant that the complex remain soluble in aqueous environments. Toensure that no precipitation of amphotericin B would occur in an aqueousenvironment, aliquot B was added dropwise to a 5% dextrose solutionwhich is similar to plasma in osmolality. No precipitate resulted.

For purpose of comparison, amphotericin B dissolved in DMSO was added toa 5% dextrose solution. An immediate precipitate formed. Therefore,although DMSO solubilizes amphotericin B, such a solution cannot be usedin vivo because the DMSO-solubilized amphotericin B will precipitate outin plasma.

7.3. REDUCED TOXICITY OF AMPHOTERICIN B LIGAND PREPARATIONS

The toxicity of the amphotericin B preparations were evaluated in mice.

7.3.1. TOXICITY OF PEG-CHOLESTEROL

Six male Swiss-Webster mice weighing 27 gms each were given a singleintravenous injection of 400 mg/kg PEG-cholesterol. No overt signs oftoxicity were demonstrated by the mice over a one-month period of visualobservation.

7.3.2. COMPARISON OF TOXICITY OF AMPHOTERICIN B AND AMPHOTERICINB/PEG-CHOLESTEROL

Solutions of Fungizone and solutions of AmB/PEG-cholesterol wereprepared such that the concentrations of amphotericin B ranged from0.625 mg/ml to 1.25 mg/ml. These preparations were inoculatedintravenously (in approximately 0.2 ml injections) in male Swiss Webstermice (average weight approximately 35 g). The animals were observed forsurvival. The percent mortality was plotted against the log dose todetermine the LD₅₀ for Fungizone and for the AmB/PEG-cholesterolpreparations. The LD₅₀ of Fungizone was 3.8 mg/kg, whereas the LD₅₀ ofAmB/PEG-cholesterol was 10.0 mg/kg. Therefore, the AmB/PEG-cholesterolpreparation is less toxic than the Fungizone. In fact when compared toFungizone, the effective dose of the amphotericin B can be increasedabout 2.5 times when the amphotericin B is administered with thePEG-cholesterol.

7.3.3. TOXICITY OF VARIOUS AMPHOTERICIN B PREPARATIONS IN MICE

To determine the toxicities of various amphotericin B preparations inmice, the following solutions were prepared in distilled water andadministered intravenously in mice:

(1) 7 mg/ml of AmB plus 50 mg/ml of PEG-cholesterol (PEG-Chol);

(2) 7 mg/ml AmB plus 7 mg/ml deoxycholate (DOC);

(3) 7 mg/ml AmB plus 7 mg/ml DOC plus 9 mg/ml PEG-Chol; and

(4) 7 mg/ml DOC.

Each solution was inoculated into the tail vein of female adult SwissWebster mice (in groups of four mice, weight 23 g each). The animalswere then observed for appearance of blue tail which may indicate abreakdown of the complex, resulting in the precipitation of amphotericinB and subsequent blockage of the blood vessel.

The results of the treatment are shown in Table V.

                  TABLE V                                                         ______________________________________                                        CONDITIONS OF MICE FOLLOWING ADMINISTRATION                                   OF VARIOUS AMPHOTERICIN B PREPARATIONS                                                                   Appearance of Blue                                                            Tail in Each Group                                 Group  Inoculum            of 4 Mice.sup.1                                    ______________________________________                                        1      AmB + PEG--Chol     ----                                               2      AmB + DOC           ++++                                               3      AmB + DOC + PEG--Chol                                                                             ---+                                               4      DOC                 ---*                                               ______________________________________                                         .sup.1 + designates one mouse with discoloration extending the length of      the tail.                                                                     - designates one mouse with no tail discoloration.                            * designates one mouse with discoloration limited to the tip of the tail.

The appearance of blue tail in the mice is evidence of precipitatedmaterial clogging an artery in the tail. Thus, in Group 1, the totalabsence of blue color in tails indicates that a solution of amphotericinB and PEG-cholesterol forms a stable complex; i.e., one which does notprecipitate out of serum. For group 2, blue tails in all the miceindicate that a solution of amphotericin B and deoxycholate precipitatesout of sera and blocks the tail artery. A lower incidence of blue tailsin Group 3 indicates that PEG-cholesterol lessens the amount ofprecipitation of an AmB-DOC solution. Finally, deoxycholate by itself(as shown for Group 4) is responsible for a limited blue tail effect.

Thus, the results in Table V tend to show that the mechanism of toxicitycould be mediated through binding in situ to low affinity lipid toxicityreceptors in the tail and that this toxicity can be attenuated (as shownfor Group 1) by preincubation in vitro of the drug with its toxicityreceptor.

7.3.4. REDUCED TOXICITY OF AMPHOTERICIN B PREPARATIONS IN CELL CULTURE

Five preparations of amphotericin B were studied to determine the effecteach preparation had on cell growth. The preparations were as follows:

1) AmB/PEG-Chol (1:3.9), (AmB:PEG-cholesterol in a 1:3.9 ratio);

2) AmB/PEG-Chol (1:14), (AmB:PEG-cholesterol in a 1:14 ratio);

3) AmB plus DOC (Fungizone);

4) PEG-cholesterol;

5) DOC.

The minimum inhibitory concentration (M.I.C.) of the amphotericin Bpreparations for C. albicans was determined as follows: each of the 5preparations was serially diluted in a 96 round-bottomed well plate,using 1/2 step dilutions of drug in 50 μl Mico-broth per well and 3wells for each dilution step. After dilutions were completed, each wellreceived 50 μl broth containing 10³ cells of C. albicans. Plates wereincubated overnight at 35° C. in a humidified atmosphere. The endpointof M.I.C. was determined as the maximum dilution of the drug showing 50%growth inhibition in comparison with control well cultures containing nodrug, and, therefore, demonstrating 100% growth.

Cytotoxicity for L-cells was also determined in 96 flat-well plates. Thedrug was diluted serially (1/2 step dilutions) in 50 μl of Eagle'sMinimum Essential Medium (MEM) containing 10% fetal bovine serum. Theneach well received a 50 μl aliquot from a suspension of 3×10⁵ L-cells/mlmedium. After incubation for 72 hours at 37° C. in a humidifiedatmosphere of 5% CO₂ in air, the L-cell monolayers were fixed with 5%formaldehyde and stained with 0.02% crystal violet in 5% formaldehyde.The endpoint was determined as the maximum dilution of the drug showing50% growth inhibition in comparison with the control well cultures (nodrug, 100% growth). The results of these experiments are shown in TableVI.

                  TABLE VI                                                        ______________________________________                                        INHIBITION OF CELL GROWTH BY DIFFERENT                                        PREPARATIONS OF AMPHOTERICIN B                                                                  Minimum Inhibitory                                                            Concentration of                                                              Amphotericin B (μg/ml)                                   Preparation         C. albicans                                                                             L cells                                         ______________________________________                                        (1)  AmB:PEG--Chol (1:3.9)                                                                            0.2       166.6                                       (2)  AmB:PEG--Chol (1:14)                                                                             0.2       166.6                                       (3)  AmB plus DOC (Fungizone)                                                                         0.4        62.5                                       (4)  PEG-Cholesterol    25,000+    500.0+                                     (5)  DOC                 5,000+   250.0                                       ______________________________________                                    

The results in Table VI demonstrate that the MIC for theAmB/PEG-cholesterol preparations is approximately 2.7 times greater thanthe MIC for Fungizone, i.e., Fungizone inhibits cell growth at lowerconcentrations than AmB/PEG-cholesterol. Therefore, theAmB/PEG-cholesterol preparations are less cytotoxic than Fungizone.

Plastic petri dishes (35 mm diameter) were seeded with 50 L-cells perdish in 2 ml MEM plus 10% fetal calf serum containing one of theamphotericin B preparations (2 dishes per amphotericin B preparation).The dishes were incubated for 10 days and the cloned cultures werestained with crystal violet as described above. The results are shown inTable VII.

                  TABLE VII                                                       ______________________________________                                        EFFECT OF DIFFERENT AmB PREPARATIONS                                          ON CLONING EFFICIENCY OF L CELLS                                                        Number of Clones                                                                           Size of Colonies                                                   Number of % of     Mean   % of                                    Preparation.sup.1                                                                         Colonies  Control  Diameter                                                                             Control                                 ______________________________________                                        AmB/PEG--Chol                                                                             75        78.5     1.8    72                                      (1:3.9)                                                                       AmB/PEG--Chol                                                                             66        68.8     1.8    72                                      (1:14)                                                                        AmB plus DOC                                                                              53        55.2     1.5    60                                      (Fungizone)                                                                   DOC         68        70.8     2.3    92                                      PEG--Chol   83        86.5     2.5    100                                     Control     96        100.0    2.5    100.0                                   ______________________________________                                         .sup.1 All three preparations of AmB were added to a final concentration      of 25 μg amphotericin B/ml medium.                                    

The results in Table VII also confirm that the AmB/PEG-cholesterolpreparations are less cytotoxic than Fungizone.

7.3.5. COMPARISON OF HEMOLYTIC PROPERTIES OF AMPHOTERICIN B PREPARATIONS

The commerical Fungizone preparation causes a significant lysis of redblood cells whereas the amphotericin B/PEG-cholesterol preparations ofthe present invention do not. This property makes the amphotericinB/PEG-cholesterol preparations more suitable for use in vivo. Details ofthe experiments are described below.

Equal aliquots of a final suspension of 2% fresh erythrocytes were mixedwith serial dilutions of either Fungizone (i.e., amphotericin B withdeoxycholate), amphotericin B/PEG-cholesterol (prepared as previouslydescribed), PEG-cholesterol, or deoxycholate in phosphate bufferedsaline free of Ca⁺⁺ and Mg⁺⁺ ; the mixtures were incubated at 37° C. for20 hours after which the cells were pelleted by centrifugation and thereleased hemoglobin in the supernatants was determinedspectrophotometrically at an OD_(550nm). Results are presented in TableVIII.

The results in Table VIII clearly demonstrate that theAmB/PEG-cholesterol preparation is less hemolytic than equalconcentrations of amphotericin B as a Fungizone preparation.

                  TABLE VIII                                                      ______________________________________                                        HEMOLYTIC EFFECT OF                                                           DIFFERENT PREPARATIONS OF AMPHOTERICIN B                                      Concentra-                                                                            OD.sub.550 nm                                                         tion.sup.1        AmB/                                                        (μg/ml)                                                                            AmB/DOC   PEG--Chol  DOC    PEG--Chol                                 ______________________________________                                        0       0.00      0.00       0.00   0.00                                      1       0.25      0.03       0.03   0.03                                      2       0.88      0.05       0.03   0.03                                      2.5     0.28      0.06       0.03   0.03                                      15      1.5+      0.24       0.03   0.03                                      30      1.5+      0.44       0.03   0.03                                      60      1.5+      0.90       0.06   0.03                                      125     1.5+      1.16       0.23   0.03                                      250     1.5+      1.38       0.47   0.03                                      ______________________________________                                         .sup.1 The concentration refers to the concentration of AmB when present      in the preparation (i.e., in AmB/DOC, and AmB/PEG--Chol preparations).        Concentration of the preparations containing no amphotericin B (i.e., DOC     and PEG--Chol) refers to the concentration of the PEGCholesterol or           deoxycholate in equivalents.                                             

We claim:
 1. A drug preparation of reduced toxicity, comprising: amixture of (a) a pharmaceutically active therapeutic drug which exhibitstoxic effects in humans or animals by binding to a toxicity receptor,the drug being selected from the group consisting of the polyene orpolyene macrolide antibiotics, anthracyclines, and cisplatin, and (b) aligand capable of preventing binding of the drug to the toxicityreceptor in vivo while allowing the drug to retain its pharmaceuticalactivity, the ligand being a phospholipid when the drug is ananthracycline or cisplatin, and the ligand being a sterol or watersoluble derivative thereof when the drug is a polyene macrolideantibiotic, wherein the drug to ligand ratio reduces the toxicity of thedrug in vivo and wherein the preparation is substantially free ofliposomes.
 2. The drug preparation according to claim 1 wherein themixture is a solution.
 3. The drug preparation according to claim 1,wherein the pharmaceutically active therapeutic drug is lipophilic. 4.The drug preparation according to claim 1 wherein the water solublesterol is polyethylene glycol-cholesterol.
 5. The drug preparationaccording to claim 1 wherein the drug is a polyene or polyene macrolideantibiotic and the ligand is a sterol or water soluble derivativethereof capable of blocking binding of said polyene or polyene macrolideantibiotic to its toxicity receptor.
 6. The drug preparation accordingto claim 5 wherein the water-soluble sterol is a water solublecholesterol.
 7. The drug preparation according to claim 6 wherein thewater soluble cholesterol is polyethylene glycol-cholesterol.
 8. Thedrug preparation according to claim 6 wherein the water-solublecholesterol is cholesterol hemisuccinate.
 9. The drug preparation ofclaim 5 wherein the mole ratio of antibiotic to ligand is from about 1:1to about 1:14.
 10. The drug preparation of claim 9 wherein the moleratio of antibiotic to ligand is about 1:1.
 11. The drug preparation ofclaim 10 wherein the sterol is cholesterol.
 12. The drug preparation ofclaim 9 wherein the mole ratio of antibiotic to ligand is about 1:5. 13.The drug preparation of claim 9 wherein the mole ratio of antibiotic toligand 1:5.9.
 14. The drug preparation of claim 9 wherein the mole ratioof antibiotic to ligand is about 1:1.6.
 15. The drug preparation ofclaims 5 or 9 wherein the antibiotic is amphotericin B.
 16. The drugpreparation of claim 15 wherein the sterol is cholesterol.
 17. The drugpreparation according to claim 5, wherein said polyene macrolideantibiotic is amphotericin B or a pharmaceutically active salt thereof.18. The drug preparation of reduced toxicity of claim 5, wherein saidligand is at a concentration below the concentration needed to formliposomes.
 19. The drug preparation of claim 18 wherein the sterol orwater soluble derivative thereof is cholesterol.
 20. The drugpreparation of claim 19 wherein the polyene or polyene macrolideantibiotic is amphotericin B.
 21. The drug preparation of claim 20wherein the mole ratio of amphotericin B to the sterol or water solublederivative thereof as ligand is from 1:1 to about 1:14.
 22. The drugpreparation of claim 21 wherein the mole ratio of amphotericin B isabout 1:1.
 23. The drug preparation of claim 22 wherein the mole ratioof amphotericin B is about 1:5.
 24. The drug preparation according toclaim 1, wherein the drug is cisplatin, and the ligand is a phosphateester, phosphate anhydride or sulfatide capable of blocking binding ofcisplatin to its toxicity receptor.
 25. The drug preparation accordingto claim 24, wherein said ligand is lysophosphatidylcholine.
 26. Thedrug preparation according to claim 24, wherein said ligand isphosphorylcholine.
 27. The drug preparation according to claim 24,wherein said ligand is phosphorylserine.
 28. The drug preparationaccording to claim 24, wherein said ligand is phosphorylglyceric acid.29. The drug preparation according to claim 24, wherein said ligand isinositol monophosphate.
 30. The drug preparation according to claim 24,wherein said ligand is inositol biphosphate.
 31. The drug preparationaccording to claim 24, wherein said ligand is inositol triphosphate. 32.The drug preparation according to claim 24, wherein said ligand isinositol tetraphosphate.
 33. The drug preparation according to claim 24,wherein said ligand is inositol pentaphosphate.
 34. The drug preparationaccording to claim 24, wherein said ligand is inositol hexaphosphate.35. The drug preparation according to claim 24, wherein said ligand is anucleotide.
 36. The drug preparation according to claim 24, wherein saidligand is phosphatidylinositol phosphate.
 37. The drug preparationaccording to claim 24, wherein said ligand is phosphatidylinositolbisphosphate.
 38. The drug preparation according to claim 24, whereinsaid ligand is phosphorylinositol.
 39. The drug preparation according toclaim 24, wherein the drug is triphosphorylinositol.
 40. The drugpreparation according to claim 1, wherein the drug is an anthracyclineand the ligand is cardiolipin, cardiolipin head group,phosphatidylserine, phosphorylserine, phosphatidic acid ortripolyphosphate, wherein the preparation is substantially free ofliposomes.
 41. A drug preparation of reduced toxicity comprising apolyene or polyene macrolide antibiotic and a ligand wherein the ligandis a sterol or water soluble derivative thereof.
 42. The method foradministering a drug preparation of reduced toxicity, comprising:administering to an animal or human a therapeutically effective amountof a solution of claim
 2. 43. A method for safely administering apolyene or polyene macrolide antibiotic for the treatment of fungalinfections, comprising: administering to an animal or human having afungal infection a therapeutically effective amount of a drugpreparation, comprising a mixture of (a) a polyene or polyene macrolideantibiotic and (b) a cholesterol or sterol or a water soluble derivativethereof.
 44. The method according to claim 43, wherein the polyenemacrolide antibiotic is amphotericin B.